Ubiquitination is a signal in diverse biological processes, including endocytosis, intracellular protein trafficking, DNA repair, kinase activation, and proteasomal proteolysis. These multiple consequences of ubiquitin (Ub) tagging indicate that there are mechanisms to discriminate among different Ub-conjugated proteins. A central hypothesis to be tested in this research is that differential outcomes can rely, in part, on the assembly of Ub into different types of polyUb chains (involving different Lys residues of Ub), which are then subject to recognition by distinct factors. The proposed research tests this hypothesis through analyses of the proteasomal recognition and processing of substrates carrying two different types of polyUb chains (linked through Lys-48 or Lys-29) because our preliminary results suggest that these two types of chains may be productively, but differentially, recognized by proteasomes. We will also compare the proteasomal degradation of polyubiquitinated substrates that differ only in the presence or absence of a ubiquitin moiety that is linearly fused to the substrate, in order to address a) how the need to unfold ubiquitin influences the rate and pathway of proteasome-dependent degradation and b) how an extraproteasomal chaperone complex containing Cdc48/VCP functions in the degradation of ubiquitin-fused substrates. Proteasome inhibitors are currently in late-stage clinical trials for the treatment of certain cancers. From the results of our research will come an improved understanding of the basic functioning of 26S proteasomes, and ultimately, the possibility of improved strategies for inhibitor design. Finally, we will study the molecular determinants of catalysis and specificity in the assembly of different polyUb chains as catalyzed by a single HECT domain Ub ligase (E3). If these studies are successful, they will provide important new information about the molecular mechanisms by which ubqiuitination is translated into specific downstream events. The results of this research will also shed new light on the mechanism of the 26S proteasome, which is the major entity for regulatory proteolysis in eukaryotic cells.